Method for producing human anti-thymocyte immunoglobulins

ABSTRACT

Methods for producing improved anti-human thymocyte immunoglobulins from specific-pathogen-free animals are provided, without the need for an adsorption step on human tissues and the consequent drawbacks of such a step.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for producinganti-human thymocyte immunoglobulins.

BACKGROUND OF THE INVENTION

[0002] Anti-human thymocyte antibodies or immunoglobulins (Igs) areknown to be selective immunosuppressants. They act on the immuneresponse by decreasing, by depletion, according to various mechanisms,the quantity of circulating lymphocytes of the blood and of the variouslymphoid tissues, and probably by blocking or modulating theirreceptors. Their use is indicated in the context of organ transplants,for the prevention and treatment of transplant rejection, and also forthe treatment of acute graft versus host reaction. These immunoglobulinshave also been proposed in the treatment of medullary aplasia.

[0003] Preparations of this type of immunoglobulin were initiallyobtained by injection of lymphatic cells into animals, rabbits or horsesfor example, and then by extraction of the anti-thymocyteimmunoglobulins from the immune serum. However, the level of purity ofthese preparations was insufficient to render these immunoglobulinsusable, in particular in therapy (R. D Guttman, 1967, J. Exp. Med.,126:1099-1127). Antihuman erythrocyte or anti-basal membraneimmunoglobulins were in particular produced by the animal during theimmunization. Several hypotheses explain the appearance of theseimmunoglobulins: either the thymocyte suspensions contain traces of redblood cells (J. M. Rolland, 1972, Pathology, 4(2): 85-122), or commonsurface antigens exist between T cells, B cells and erythrocytes, whichat the current time is the most plausible hypothesis.

[0004] Techniques were then developed in order to improve the purity ofthe preparation. These techniques envisioned a step of hemadsorption,namely adsorption on human red blood cells.

[0005] This step of hemadsorption makes it possible to remove themajority of the undesirable antibodies, such as the anti-erythrocyteantibodies present in the initial immune serum. A step of removal of theanti-tissue antibodies, on human tissues (such as placenta), was alsosometimes envisaged, in order to adsorb the anti-basal membraneantibodies in particular. Several anti-human thymocyte immunoglobulinsprepared according to these techniques are now available on the market,such as Thymoglobuline® (Imtix-Sangstat), Tecelac® (Biotest), or elseATGF® (Fresenius) or ATGAM (Upjohn).

[0006] However, the methods described which comprise a step ofadsorption on human tissues or red blood cells still exhibit drawbacks.In particular, in carrying out these methods, a 30% decrease in thelevel of gammaglobulins contained in the serum, and therefore a notinsignificant part of the antibodies of interest, is observed. Inaddition, the removal of the undesirable anti-erythrocyte antibodies isnot complete. Now, these antibodies would be liable to cause theappearance of hemolytic anemia in treated patients. Finally, the use ofhuman material, such as erythrocytes, even purified and made safe, inthe method for isolating the anti-human thymocyte immunoglobulins,increases the potential risk of infection.

[0007] The authors of the present invention have therefore sought todevelop an improved method for producing anti-human thymocyteimmunoglobulins.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a method with no step ofadsorption on human biological material, for producing a preparation ofanti-thymocyte immunoglobulins which is sufficiently pure for use intherapy in particular.

[0009] More precisely, a subject of the invention is a method forproducing anti-purified human thymocyte immunoglobulins, which comprisesthe steps consisting in:

[0010] (a) injecting a cellular preparation of human thymocytes into aspecific-pathogen-free (SPF) animal;

[0011] (b) collecting the immune serum produced by the animal;

[0012] (c) isolating the anti-human thymocyte immunoglobulins from theserum, with no adsorption on human biological material.

[0013] More particularly, the expression “immunoglobulins from serum” isintended to mean the total fraction of gammaglobulins from the serumafter immunization.

[0014] The expression “adsorption on human biological material” isintended to mean in particular hemadsorption on red blood cells oradsorption on human tissues (such as placenta in particular), stroma orcrude extracts of these tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The SPF animals are breeding animals whose environment and foodare strictly controlled according to health standards. Reference is alsomade to animals “with controlled health status”. The SPF animals arebred in closed, sterilized compartments, the air environment beingfiltered, for example through a HEPA filter (sterilizing filtration),and the water and foodstuff being decontaminated before introduction,which makes it possible to eliminate any pathogenic element from theirdirect environment (Yanabe et al. Exp. Animal 48(2), 101-106 (1999)).The term “pathogen” here denotes an infectious agent capable of causinga clinical disease and/or of modifying the biological response of theanimal with regard to the desired use. The SPF status refers to a list(which evolves) of microorganisms and of methods of control (clinical,serological, histological, or using culturing) for detecting thetargeted microorganisms or, conversely, demonstrating their absence.

[0016] Use is preferentially made of horses or goats, more preferablyrabbits, for the production of immune serum. In fact, only IgG1s arepresent in rabbit serum, which facilitates the process of antibodypurification. Rabbit IgG also shows a high affinity for the human Fcreceptor, allowing the development of powerful cytotoxic antibodiesdirected against human T cells.

[0017] The cellular preparation of human thymuses can be obtained eitherfrom cells of lines in culture, or from fresh thymocytes which arepurified preferentially from human thymus fragments or optionally from,for example, suspensions of spleen, tonsils, lymph nodes, thoracictrachea or peripheral blood. The human thymus fragments can inparticular be easily removed during surgical acts, in particularsubsequent to cardiac surgery on children. Virological tests are carriedout on the donor's blood in order to avoid any contamination and toeliminate any contaminated thymic fragment showing a positiveserological result.

[0018] The injecting of the human thymocytes and the collecting of theimmune serum from the SPF animal are carried out according to standardtechniques of those skilled in the art.

[0019] Isolation of the anti-human thymocyte immunoglobulins from theserum makes it possible to eliminate the undesirable proteins. It can becarried out using, for example, ion exchange chromatography, preferablyon a column, and/or one or more precipitations.

[0020] Advantageously, such a precipitation can be carried out in twosuccessive steps using an immunoglobulin precipitating reagent. Thereagent preferentially used is sodium sulfate.

[0021] The chromatography step enables retention of the loadedimpurities by an ion exchange resin such as anions (DEAF). As regardsthe IgGs, they are not retained by the column and are rapidly removedfrom the column, which makes it possible to harvest them selectively.Chromatography on a specific affinity column, which removes theremaining undesirable antibodies, is also advantageous.

[0022] A subject of the invention is also the isolated antihumanthymocyte immunoglobulins which can be prepared using the method of theinvention.

[0023] These immunoglobulins can also be designated “antihumanlymphocyte immunoglobulins” since they recognize human lymphocytes whenthey are brought into contact with these lymphocytes, for example wheninjected into patients for the purpose of immunodepletion.

[0024] Moreover, the present invention is also directed toward the useof these immunoglobulins for producing a medicinal product intended todecrease the quantity of circulating lymphocytes of the blood and of thelymphoid tissues in a human patient.

[0025] A subject of the invention is also a method of therapeutictreatment in which a therapeutically effective quantity ofimmunoglobulins thus obtained is administered to a patient requiring hisor her quantity of lymphocytes to be decreased. These immunoglobulinsare particularly useful in the context of organ transplants.

[0026] The anti-human thymocyte immunoglobulins prepared according tothe method described above exhibit a greater specific activity than theanti-thymocyte immunoglobulins prepared using a method including ahemadsorption step, as emerges in Example 2.1 presented below.

[0027] Deleting the step of adsorption on human biological materialmakes it possible to produce immunoglobulins without any contact with ahuman derivative. This leads to the elimination of any possible viralcontamination or contamination with unconventional agents of the priontype. In addition, any contamination associated with hemadsorption,namely contamination with the hemoglobin released by the human red bloodcells during the hemolysis engendered by this step, is avoided.

[0028] The method of the invention makes it possible to obtain apreparation of anti-thymocyte immunoglobulins in which the number ofimmunoglobulins capable of cross reacting with other blood cells(erythrocytes, neutrophils, etc.) is considerably reduced. In addition,the fact that hemadsorption is not used in preparing the anti-thymocyteantibodies makes it possible not only to improve the safety aspect, butalso reduces the length and the cost of the preparation method. In fact,hemadsorption conventionally uses whole human red blood cells which arefresh and formalin-treated, and requires large quantities of cells to betreated, making the production of antithymocyte immunoglobulinsrelatively restrictive in industrial terms.

[0029] The following examples illustrate the invention without limitingthe scope thereof.

EXAMPLES Example 1 Production of Immunoglobulins

[0030] 1.1 Purification of Thymocytes

[0031] Human thymus fragments are, after removal and grinding, filteredand placed in suspension.

[0032] The cell suspension is then filtered through a nylon cloth andsubjected to centrifugation at 1800 rpm at 5° C.

[0033] 20 ml of Ficoll are added to 10 ml of cell solution containing 2to 7×10⁹ cells and the mixture is centrifuged at 2000 rpm at 5° C. Twosubsequent centrifugations are carried out at 1800 rpm. The cellularpreparations are then stored at 5° C. overnight and are then dilutedbefore being injected into SPF rabbits. The thymocytes can also beconserved by freezing.

[0034] 1.2 Isolation of Antibodies:

[0035] The rabbit immune serum is collected in the course of severalbleeds between D20 and D30, and can be frozen for storage. During thepreparation, batches of serum are formed which are gradually returned toambient temperature.

[0036] These batches of rabbit serum are decomplemented in order toremove the complement proteins, by bringing them to a temperature of56□C±2□C for a period of 30 to 45 min.

[0037] The rabbit serum is purified for the immunglobulin fraction bychromatography on an anion exchange resin (DEAE) at ambient temperaturefollowed by two precipitations with sodium sulfate.

[0038] After a further step of filtration, concentration anddiafiltration, the anti-thymocyte immunoglobulins are pasteurized at atemperature of 60° C. for 10 hours in order to ensure the viral safetythereof.

[0039] According to one variant, the steps of filtration, concentration,diafiltration and precipitation of the immunoglobulin fraction (forexample alcoholic fractionation of the COHN type or ammonium sulfatefractionation) can be carried out before the chromatography step.

[0040] The solution of immunoglobulins can, after formulation andsterilization by filtration, be conserved in the liquid state in asolution of 5 to 25 mg/ml or, according to one variant, in thelyophilized state. A series of quality controls is executed, includingphysicochemical tests, safety tests (presence of pyrogenic agents, ofantiplatelet activity), sterility and purity tests and alsolymphocytotoxic activity tests (inhibition of rosette formation in vitroand allogenic skin graft survival in monkeys).

Example 2 Comparative Analysis of the Effectiveness and the Innocuity ofthe Immunoglobulins Obtained Using the Method of the Invention, withRespect to Conventional Immunoglobulins (Obtained with Hemadsorption)

[0041] 3.1 Measurement of the Specific Activity In Vitro

[0042] Two batches of immunoglobulins were used for the comparativestudy of the specific activity: a standard anti-thymocyte immunoglobulin(ATG) batch and a nonhemadsorbed ATG batch.

[0043] To determine the specific activity of the ATGs, i.e. to evaluatethe quantity of immunoglobulins capable of binding to human lymphocytes,a technique of flow cytometry coupled to indirect immunofluorescence isused. Whole monkey blood (containing all blood cells) is incubated withthe ATG batches at various concentrations. The excess of unattachedimmunoglobulins is rinsed off and a second antibody, labeled withfluorescein isothiocyanate (FITC), is added so as to attach to the boundATGs. The amount of binding of the ATGs to the surface of the cells isthen revealed after differentiation of the cells as a function of theirsize using flow cytometry.

[0044] The results show a greater specific activity of thenon-hemadsorbed ATGs. Thus, to obtain the same binding to lymphocytes,2.73 μg/ml of non-hemadsorbed ATG are necessary, where 7.23 μg/ml ofstandard ATG are necessary, i.e. 2.65 times more. The hemadsorption steptherefore decreases the binding capacity and the specific activity ofthe ATGs.

[0045] 3.2 Measurement of the Activity of the ATGs in Vivo in the SkinGraft Test in Monkeys

[0046] Three groups of monkeys were selected for carrying out this test:a control group (n=5), a group receiving 5 mg/kg of standard ATG (n 8)and a group receiving 5 mg/kg of ATG not hemadsorbed (n=3) initially. A50/50 dilution of the non-hemadsorbed ATGs is carried out in order toobtain a specific activity equivalent to that of the standard batch.

[0047] The skin graft test consists in performing, on DO, fourallografts (originating from an animal in which at least two of the HLAantigenic determinants are different) and an autograft of skin on theback of each animal. The doses of ATG are then administered in vivo forthree weeks from D-1.

[0048] The results show that the mean survival times of the allograftsare equivalent for the two groups of treated monkeys: 23 days (±4.16)for the standard ATG group and 21.3 days (±7.3) for the non-hemadsorbedATG group. They are also greater than the control: 9.4 days (±1.5).

[0049] The prolonging of the survival time of the skin graft correlateswith the lymphocyte depletion induced by the administration of ATG. Thelymphopenia follows an evolution which is comparable between the twobatches: maximum decrease in the level of lymphocytes at D5, thengradual return to normal values around D20.

[0050] Consequently, the diluted batch of non-hemadsorbed ATGs isequivalent, in terms of effective in vivo in the primate, to theundiluted standard ATG batch.

[0051] 3.3 Evaluation of the Risks of Hemolytic Anemia Subsequent toAdministration of ATGs

[0052] In the context of the same study, blood tests are carried outregularly until the end of the test and show that the evolution of thehemoglobin level is identical in the two groups of treated monkeys. Adecrease in this level is observed up to D9, until this level graduallyreturns to normal values. Two phenomena are responsible for thisevolution: firstly, the anemia is of the iatrogenic type, subsequent tothe repeated taking of blood samples. This anemia is identical in thethree groups of monkeys. Secondly, an effect specific to the ATGs causesa substantial decrease in the hemoglobin level from D1 in the treatedgroups, this decrease being identical with the standard ATG or with thenon-hemadsorbed ATG.

[0053] In addition, in order to detect a possible hemolytic cause forthe anemia, the levels of haptoglobin and of oromucoid are alsomeasured. In the event of hemolysis, haptoglobin becomes unmeasurable.Now, these two levels increase from D4 for the two groups, this increasereflecting an inflammatory activity.

[0054] Thus, the hematologic safety evaluated in the animals treatedwith the non-hemadsorbed ATG is as satisfactory as in the animalstreated with the hemadsorbed ATG.

1. A method for producing anti-human thymocyte immunoglobulins,comprising the steps of: (a) injecting a cellular preparation of humanthymocytes into a specific-pathogen-free (SPF) animal; (b) collectingthe immune serum produced by the animal; (c) isolating the anti-humanthymocyte immunoglobulins from the collected immune serum, with noadsorption on human biological material.
 2. The method according toclaim 1, wherein the SPF animal is a rabbit.
 3. The method according toclaim 1, wherein the isolation step (c) comprises a chromatography step.4. The method according to claim 3, wherein the chromatography stepcomprises chromatography on an ion exchange resin on a column.
 5. Themethod according to claim 4, wherein said ion exchange resin is an anionexchange resin.
 6. The method according to claim 5, wherein said anionexchange resin is DEAE.
 7. The method according to claim 3, wherein thechromatography step further comprises chromatography on a specificaffinity column.
 8. The method according to claim 3, wherein saidisolation step (c) further comprises at least one precipitation step. 9.The method according to claim 8, wherein said at least one precipitationstep uses an immunoglobulin-precipitating reagent.
 10. The methodaccording to claim 9, wherein said immunoglobulin-precipitating reagentis sodium sulfate.
 11. The method according to claim 1, wherein thecellular preparation of human thymocytes used in step (a) consists ofcells of lines in culture.
 12. The method according to claim 1, whereinthe cellular preparation of human thymocytes used in step (a) isobtained by purification of fresh human thymocytes.
 13. An isolatedanti-human thymocyte immunoglobulin which can be prepared using themethod as claimed in any one of claims 1 to
 12. 14. A method fordecreasing the quantity of circulating lymphocytes of the blood and ofthe lymphoid tissues in a human patient, which comprises administratingan effective amount of an immunoglobulin of claim 13 to said patient.